The long-term objective of this proposal is to elucidate the molecular mechanisms controlling the formation of a functional metanephric mesenchyme during early kidney development by examining the role of the transcriptional coactivator Eya1, its interacting homeodomain protein Six1 and their cofactors. The mammalian kidney develops in a region of posterior intermediate mesoderm by inductive interactions between the metanephric mesenchyme and the ureteric bud epithelium. Recent genetic and molecular studies have indicated that the metanephric mesenchyme may provide initial signals to promote ureteric bud formation. However, what genes and the regulatory hierarchy controlling the formation of the metanephric mesenchyme still remain unclear. Recently, the murine Eya1 gene was found to be expressed in the metanephric mesenchyme and mutations in the human EYA1 gene cause Branchio-Oto-Renal (BOR) syndrome, a congenital birth defect characterized by combinations of branchial, otic and renal anomalies. However, despite the identification of the responsible gene, the developmental and molecular basis for renal defects and the identity of the steps at which Eya1 functions in early kidney morphogenesis are unclear. In Eya1 -/- mice, the metanephric mesenchyme never forms and the mesenchymal cells undergo abnormal apoptosis from E10.5, indicating that Eya1 is a key mesenchymal gene required for early kidney morphogenesis. The homeobox gene Six1 was also found to play an essential role during early kidney development and its gene product physically interacts with Eya1. Moreover, the transcription factor N-myc has been recently isolated through yeast two-hybrid screen using Eya1 as "bait" and it physically interacts with both Six1 and Eya1 in vitro. Interestingly, mutations in the N-myc gene also cause kidney defects. Based on these data, we hypothesize that these transcription factors function together in the metanephric mesenchyme to regulate early kidney development. This grant will use a powerful genetic system to test this hypothesis and integrate several mesenchymal genes into a genetic and molecular regulatory pathway governing early kidney development. Specifically, I propose to: (1) test the functional role of Eya1 and Six1 during early kidney development, (2) test the possible interactions between Six1, Eya1 and Pax2 in early kidney development, (3) test whether N-myc interacts with Eya1 or Six1 during early kidney development. These studies will clarify the relationship between Pax2, Eya1, Six1, N-myc and other genes, and have a strong likelihood of providing significant insight at the molecular and genetic level into the early developmental process of kidney morphogenesis.